Fig. 7: Overexpressing USP25 in IgG4-RD cell line rescues the clinical phenotype of IgG4-RD patients.

A–E TIRFm analysis of the MFI of F-actin, ALDOA and the correlation coefficient between F-actin and ALDOA (scale bar = 2.5 μm). F, G PBMCs and splenic B cells were pre-incubated with sAg for 24 h and then permeabilized with 30 μg/ml of digitonin for 5 min and the protein levels of ALDOA in the supernatant and lysates were analyzed. H, I PBMCs were pre-incubated with 10 μM of Latrunculin B for 2 h at 37 °C and then incubated with anti-CD19, followed by stimulation with sAg. PBMCs were stained with F-actin and pWASP after fixation and permeabilisation, and analyzed (n = 3). J PBMCs were pre-incubated in the same way as in (Fig. 7H) except stained for anti-CD19 and CellIROX Green and the MFI of CellIROX Green were analyzed (n = 3). K RT-PCR was used to analyze the mRNA levels of TP1, ECON, and PKM2 in purified B cells (n = 3). L Western blot of USP25 in PBMCs and cell lines. M Cell lines were analyzed for the MFI of CellIROX Green in the same way as in (Fig. 5N). N ECAR detection of B cells from HC cell line and IgG4-RD cell line (n = 3). O Western blot of USP25, pWASP, pSYK, pBLNK, pmTOR, pS6, c-MYC, COL1A1, and NLRP3 in B cells of HC cell line and IgG4-RD cell line stimulated with sAg for designated times. P HC cell line and IgG4-RD cell line were pre-incubated with 10 µM of AZ1 for 1 h at 37 °C and then stimulated with sAg for 5 min. The protein levels of USP25, LYN, pSYK, pmTOR, pS6, pFOXO1, c-MYC, COL1A1 and RAC1 were analyzed. Q After overexpression of phage-6tag-USP25 or the empty vector in IgG4-RD cell line or HC cell line by lentiviral transfection, the protein levels of USP25, pBTK, LYN, pSYK, RAC1, PKM2, and pSMAD3 were analyzed (EV=Empty Vector). All images were representative images from 3 independent experiments. Data points in (B–E, K) are represented as Mean ± SED. Statistical significance was based on two-tailed unpaired Student’s t-test. Relevant p values are given in the graph. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.